The purpose of this proposal is to determine how intestinal epithelial cells control the signaling functions of Toll-like Receptor 5 (TLR5), an important regulator of inflammatory bowel diseases (IBDs). Recent publications suggest that localization of adaptor proteins, necessary for innate immune receptor signaling, define the sub-cellular location from which TLRs signal. In accordance with this, preliminary data generated for this proposal suggests that the adaptor protein TIRAP, which has been shown to be required for TLR5 signaling, is found exclusively on the basolateral membrane of intestinal cells. Thus, we hypothesize that TIRAP's localization influences which surface (apical or basal) signaling occurs from on intestinal cells. Based on this hypothesis, this proposal seeks to (1) determine the localization of TLR5 and TIRAP in human intestinal epithelial cells (IECs), (2) Determine the functional role of TIRAP in determining the site of TLR5 signaling, and (3) Determine the mechanism by which TIRAP is localized within IECs. To address these aims in human IECs, genome editing strategies will be employed to make the genetic mutants needed for defining these innate immune signaling pathways. Microscopic techniques will be used to visualize interactions of adaptor proteins and receptors at sites of signaling, and cell biological assays wil determine the metabolic pathways important for directing where sites of signaling occur. This research has the potential to uncover new targets for those developing therapeutics for patients suffering from IBD.